Methacrylic acid esters are used mostly as raw material for producing acrylic resins, but are also required as comonomers to be used in a variety of applications such as coatings, adhesives and resin modifiers. Examples of methods for industrially producing methacrylic acid esters are acetone cyanohydrin (ACH) methods that use acetone and hydrogen cyanide as raw materials, direct oxidation methods that use isobutylene and t-butyl alcohol as raw materials, and the like. Those chemical methods depend on fossil-based raw materials and require much energy.
From the viewpoints of global warming prevention and environmental protection, in recent years industries have focused on technologies for producing various chemical products using biomass as the carbon source so as to replace conventionally used fossil materials. Methacrylic acids and methacrylate esters are also expected to be produced from biomass material.
For example, methods are proposed for producing 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid to be used as precursors of methacrylic acids from natural products such as sugars by using naturally available microorganisms (see Patent Publications 1 and 2 and Non-patent Publication 1). Other proposed methods are for producing methacrylic acids from glucose using recombinant microorganisms that do not exist naturally but are constructed by introducing enzyme genes. However, those methods are based on combined enzymatic reactions of known reactions and what is assumed from those known reactions, and thus such reactions are not verified (see Patent Publications 3˜5). Those publications provide descriptions showing that certain enzymes for catalyzing similar catalytic reactions may also be used for enzymatic dehydration reactions of 3-hydroxyisobutyric acid or 3-hydroxyisobutyryl-CoA. In fact, enoyl-CoA hydratase catalyzes dehydration reactions in the acetone/butanol fermentation pathway. So, if such certain enzymes take the above compounds as their specific substrates, they are thought to be effective. On the other hand, enoyl-CoA hydratase in β-oxidation of fatty acids or the degradation pathway of branched-chain amino acids is an enzyme for catalyzing hydration reactions but not for catalyzing dehydration reactions.
Non-patent Publication 2 has a description showing that enoyl-CoA hydratase purified from bacteria which produce poly-3-hydroxybutyrate has activities to catalyze dehydration reactions and reverse reactions (hydration reactions) of 3-hydroxybutyl-CoA. However, it is unknown whether other enoyl-CoA hydratases catalyze such two-way reactions. Moreover, there is no report in the above prior art as to whether methacrylyl-CoA has been synthesized by methods using 3-hydroxyisobutyryl-CoA as raw material. Considering diversity of enzymes and their substrate specificity, it is still unknown whether an enzyme for catalyzing only similar reactions is capable of producing methacrylyl-CoA having a structure different from its original substrate.
Meanwhile, methacrylyl-CoA is known as an intermediate in the metabolism of valine. Also, it is known to be cytotoxic. In living organisms, methacrylyl-CoA is promptly hydrated by the activity of enoyl-CoA hydratase, and is thought to be metabolized to 3-hydroxyisobutyrate through an intermediate stage of 3-hydroxyisobutyryl-CoA.
Non-patent Publication 3 describes examples in which crotonase is used to catalyze hydration reactions from methacrylyl-CoA to 3-hydroxyisobutyryl-CoA. The publication describes that the conversion rate in such reactions is lower than in other reactions (acrylyl-CoA→hydroxy propionyl-CoA) and that the reactions have reached equilibrium. However, such reactions are hydration reactions using methacrylyl-CoA as raw material, and it is totally unknown whether dehydration reactions actually progress using 3-hydroxyisobutyryl-CoA as raw material. Moreover, Non-patent Publication 4 describes spontaneous hydration reactions of methacrylyl-CoA. However, it is totally unknown whether, under aquatic conditions where methacrylyl-CoA is spontaneously hydrated, 3-hydroxyisobutyryl-CoA is actually dehydrated to produce methacrylyl-CoA as the product of interest in the present invention.